Mutagenesis of Bacillus subtilis M-4 strain with high gossypol degradation by atmospheric and room temperature plasma and optimization of fermentation process

Abstract:

[Background] Free gossypol in cottonseed meal restricts the application of the meal as a dietary protein resource, and thus the degradation of gossypol has become a research hotspot. We previously found that Bacillus subtilis M-4 can degrade gossypol and it has been used in the industrial detoxification of cottonseed meal. [Objective] To improve the ability of M-4 to degrade gossypol in cottonseed meal and expand the application of cottonseed meal in breeding industry. [Methods] We employed atmospheric and room temperature plasma (ARTP) for mutagenesis of M-4, and preliminarily screened the forward mutants with the index of gossypol degradation rate in liquid medium. Furthermore, according to the degradation rate of gossypol in solid fermentation medium, the free gossypol-degrading ability of the forward mutants was evaluated. In addition, based on gossypol residue and degradation rate of gossypol, the solid fermentation conditions for cottonseed meal were optimized by single-factor experiment and the optimal fermentation parameters were obtained. [Results] A total of 19 forward mutants were screened out and MY-4-17 was identified to be most efficient. In solid medium, the gossypol degradation rate of MY-4-17 reached 97.15%, 2.55% higher than that of M-4. MY-4-17 showed genetic stability after 5 passages. The optimal fermentation conditions for cottonseed meal with MY-4-17 are as follows: 4% corn flour as sugar source, water content in raw material of 45%–50%, MY-4-17 dose at 1.0×10¹³ CFU/t, and fermentation duration of 13–15 days. Under such conditions, the content of gossypol in cottonseed meal was reduced from 980 mg/kg to 22 mg/kg and the degradation rate was over 97.75%. [Conclusion] The mutant screened by ARTP can effectively degrade the gossypol in cottonseed meal. The result can serve as a reference for the development and production of fermentation agent for cottonseed meal.